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Gradient Gel (gradient + gel)

Distribution by Scientific Domains
Medical Sciences60%
Life Sciences40%

Terms modified by Gradient Gel

  • gradient gel electrophoresis
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  • gradient gel electrophoresis analysis
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    Selected Abstracts

    Separation of proteins with a molecular mass difference of 2,kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: Application to the isolation of 24,kDa human growth hormone

    ELECTROPHORESIS, Issue 23 2005
    Juan J. Bustamante
    Abstract A method for separating proteins with a molecular mass difference of 2,kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24,kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2,kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24,kDa hGH. The 22 and 24,kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13,18% linear gradient gel, and a 15,10% linear inverted gradient gel. Fractions containing purified 24,kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2,kDa. [source]

    Culture-independent evidence for the persistent presence and genetic diversity of microcystin-producing Anabaena (Cyanobacteria) in the Gulf of Finland

    ENVIRONMENTAL MICROBIOLOGY, Issue 4 2009
    David P. Fewer
    Summary The late summer mass occurrences of cyanobacteria in the Baltic Sea are among the largest in the world. These blooms are rarely monotypic and are often composed of a diverse assemblage of cyanobacteria. The toxicity of the blooms is attributed to Nodularia spumigena through the production of the hepatotoxic nodularin. However, the microcystin hepatotoxins have also been reported from the Baltic Sea on a number of occasions. Recent evidence links microcystin production in the Gulf of Finland directly to the genus Anabaena. Here we developed a denaturing gradient gel electrophoresis (DGGE) method based on the mcyE microcystin synthetase gene and ndaF nodularin synthetase gene that allows the culture-independent discrimination of microcystin- and nodularin-producing cyanobacteria directly from environmental samples. We PCR-amplified microcystin and nodularin synthetase genes from environmental samples taken from the Gulf of Finland and separated them on a denaturing gradient gel using optimized conditions. Sequence analyses demonstrate that uncultured microcystin-producing Anabaena strains are genetically more diverse than previously demonstrated from cultured strains. Furthermore, our data show that microcystin-producing Anabaena are widespread in the open Gulf of Finland. Non-parametric statistical analysis suggested that salinity plays an important role in defining the distribution of microcystin-producing Anabaena. Our results indicate that microcystin-producing blooms are a persistent phenomenon in the Gulf of Finland. [source]

    MUC2, MUC5AC and MUC5B in the mucus of a patient with pseudomyxoma peritonei: Biochemical and immunohistochemical study

    PATHOLOGY INTERNATIONAL, Issue 8 2007
    Anwar S. Mall
    A 58-year-old man with a 1 year history of progressive abdominal distension underwent a laparotomy for pseudomyxoma peritonei. The mucin was identified and characterized in the present study. Approximately 6 L of crude mucus in the sol (highly viscous) and gel (semisolid) phases was obtained from the patient's peritoneal cavity. The sol material was briefly homogenized followed by slow stirring at dilutions of up to 1:10 with 6 mol/L guanidinium chloride and proteolytic inhibitors for periods of up to 48 h. Preparative and analytical gel filtration on Sepharose 2B showed some PAS-positive material eluting in the void volume accompanied by equal or larger amounts of protein in the void and included volumes of the columns. Sodium dodecylsulfate,polyacrylamide gel electrophoresis of purified mucin on a 4,20% gradient gel showed PAS-positive material on the top of the running gel and a distinct smaller-sized species of mucin of higher electrophoretic mobility with background material in between the large and small mucin. Western blot (confirmed by immunohistochemical analysis) after agarose gel electrophoresis showed the presence of MUC2, MUC5AC and MUC5B in the mucus. There was no MUC1, MUC1core or MUC6 in the tissue. Histopathological examination confirmed a mucinous appendicular adenocarcinoma. Histology showed the mucin to be predominantly of the sulfated and non-sulfated acidic type. Serine, threonine and proline comprised 21.6% of the total amino acid composition of the sample. The viscous nature of the material is due to the presence of three gel-forming mucins and possibly to its high content of protein. [source]

    Identification of RSVP14 and RSVP20 Components by Two-dimensional Electrophoresis and Western-blotting

    REPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2008
    JA Cardozo
    Contents We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold-shock. In this study, we use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4,7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight (Mr) from 11.7 to 90.4. Staining of 2D gels with Pro-Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N-linked oligosaccharides improved the gel resolution. 2D-PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four (a, b, c, d) in the 14 kDa and two (e, f) in the 20 kDa region. Western-blot analyses indicated that the anti-P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a, b, c, d of F6 map. The anti-P20 antibody recognized spots 13 and 14 of SP map that corresponded to spots e, f of F6 map. The deduced sequences by de novo sequencing evidenced that protein spots 7 and 13 have significant similarities to BSP family, while protein spots 4 and 14 did not appear to be homologous with any reported protein in the current mammalian Proteinbank databases. [source]

    HDL2 of Heavy Alcohol Drinkers Enhances Cholesterol Efflux From Raw Macrophages via Phospholipid-Rich HDL2b Particles

    ALCOHOLISM, Issue 6 2008
    Sanna M. Mäkelä
    Background:, Alcohol consumption is associated with increased serum high density lipoprotein (HDL) cholesterol levels and a decreased risk for the development of atherosclerosis. However, the effects of heavy alcohol intake on reverse cholesterol transport, one of the key anti-atherogenic processes related to HDL, are poorly known. Methods:, The ability of total HDL as well as HDL2 and HDL3 subclasses to promote cholesterol efflux from 3H-cholesterol-labeled RAW 264.7 macrophages was studied among 6 heavy alcohol drinkers and 6 controls. Distribution of HDL subclasses was analyzed by 4 to 30% native gradient gels. Serum phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) activities were analyzed among several other biochemical measures. Results:, Cholesterol efflux to HDL2 of heavy drinkers was 22% (p = 0.025) higher relative to controls. The increase in HDL2 phospholipids, with a concomitant 2-fold (p = 0.055) increase in large HDL2b particles, was associated with enhanced cholesterol efflux to HDL2. Interestingly, the cholesterol efflux to HDL3 did not differ between the 2 study groups. These findings may be partially explained by a decreased CETP activity (,26%, p = 0.037) and an increased PLTP activity (39%, p = 0.045) in heavy drinkers. Conclusions:, The increased cholesterol efflux potential of HDL2 is most likely an anti-atherogenic feature linked to heavy alcohol consumption. The cholesterol efflux and HDL phospholipids also associated strongly within the whole study group (rs = 0.910, p , 0.01) suggesting a common pathway of enhanced cholesterol efflux via enlarged phospholipid-rich HDL particles. [source]